The isolation and purification of nucleic acids (DNA and RNA, for example) from complex matrices such as blood, tissue samples, bacterial cell culture media, and forensic samples is an important process in genetic research, nucleic acid probe diagnostics, forensic DNA testing, and other areas that require amplification of the nucleic acids. A variety of methods of preparing nucleic acids for amplification procedures are known in the art; however, each has its limitations.
The most common method for isolating DNA from whole blood involves the isolation of peripheral blood mononuclear cells (PBMC's) using density gradients. While this method works for research applications, it is generally not suitable for use in a conventional integrated, high throughput microfluidic device.
Hypotonic buffers containing a nonionic detergent can be used to lyse red blood cells (RBC's) as well as white blood cells (WBC's) while leaving the nuclei in tact. In another procedure, only RBC's are lysed when whole blood is subjected to freezing and thawing. The in-tact WBC's or their nuclei can be recovered by centrifugation. For lysis of RBC's without destruction of WBC's, one can also use aqueous dilution as a method. Other methods for selective lysis of RBC's include the use of ammonium chloride or quaternary ammonium salts as well as subjecting RBC's to hypotonic shock in the presence of a hypotonic buffer. However, in conventional methods using one of these approaches, substances that inhibit PCR (e.g., inhibitors of enzymes) are coprecipitated with the nuclei and/or nucleic acid. These inhibitors have to be removed prior to analysis in a conventional high throughput microfluidic device.
While treatment such as boiling, hydrolysis with proteinases, exposure to ultrasonic waves, detergents, or strong bases have been used for the extraction of DNA, alkaline extraction is among the simplest of strategies. For example, U.S. Pat. No. 5,620,852 (Lin et al.) describes an efficient extraction of DNA from whole blood performed with alkaline treatment (e.g., NaOH) at room temperature in a time frame as short as 1 minute. However, in order to get clean DNA, removal of hemoglobin as well as plasma proteins is necessary. This has been accomplished by the use of a brief washing step, for example, by suspension of the blood in water followed by centrifugation, discarding of the supernatant and then extraction of the pellet with NaOH (see, e.g., Biotechniques, Vol. 25, No. 4 (1998) page 588). The large volume of water used to lyse the cells makes the method unsuitable for use in standard microfluidic devices.
U.S. Pat. No. 5,010,183 (Kellogg et al.) describes a centrifugal microfluidics-based platform that uses alkaline lysis for DNA extraction from blood. This method involves mixing a raw sample (e.g., 5 microliters (μL) of whole blood or an E. Coli suspension) with 5 μL of 10 millimolar (mM) NaOH, heating to 95° C. for 1-2 minutes to lyse cells, releasing DNA and denaturing proteins inhibitory to PCR, neutralizing of the lysate by mixing with 5 μL of 16 mM TRIS-HCl (pH 7.5), mixing the neutralized lysate with 8-10 μL of liquid PCR reagents and primers, followed by thermal cycling. Unfortunately, while the reagent volumes are small and suitable for a microfluidic device, downstream processing of DNA in a microfluidic device is challenging.
Another conventional method uses a phenol chloroform extraction. However, this requires the use of toxic and corrosive chemicals and is not easily automated.
Solid phase extraction has also been used for nucleic acid isolation. For example, one method for isolating nucleic acids from a nucleic acid source involves mixing a suspension of silica particles with a buffered chaotropic agent, such as guanidinium thiocyanate, in a reaction vessel followed by addition of the sample. In the presence of the chaotrope, the nucleic acids are adsorbed onto the silica, which is separated from the liquid phase by centrifugation, washed with an alcohol water mix, and finally eluted using a dilute aqueous buffer. Silica solid phase extraction requires the use of the alcohol wash step to remove residual chaotrope without eluting the nucleic acid; however, great care must be taken to remove all traces of the alcohol (by heat evaporation or washing with another very volatile and flammable solvent) in order to prevent inhibition of sensitive enzymes used to amplify or modify the nucleic acid in subsequent steps. The nucleic acid is then eluted with water or an elution buffer. This bind, rinse, and elute procedure is the basis of many commercial kits, such as Qiagen (Valencia, Calif.); however, this procedure is very cumbersome and includes multiple wash steps, making it difficult to adapt to a microfluidic setting.
Ion exchange methods produce high quality nucleic acids. However, ion exchange methods result in the presence of high levels of salts that typically must be removed before the nucleic acids can be further utilized.
International Publication No. WO 01/37291 A1 (MagNA Pure) describes the use of magnetic glass particles and an isolation method in which samples are lysed by incubation with a special buffer containing a chaotropic salt and proteinase K. Glass magnetic particles are added and total nucleic acids contained in the sample are bound to their surface. Unbound substances are removed by several washing steps. Finally, purified total nucleic acid is eluted with a low salt buffer at high temperature.
Yet another conventional method involves applying a biological sample to a hydrophobic organic polymeric solid phase to selectively trap nucleic acid and subsequently remove the trapped nucleic acid with a nonionic surfactant. Another method involves treating a hydrophobic organic polymeric material with a nonionic surfactant, washing the surface, and subsequently contacting the treated solid organic polymeric material with a biological sample to reduce the amount of nucleic acid that binds to the organic polymeric solid phase. Although these solid phase methods are effective methods for isolating nucleic acid from biological samples, other methods are needed, particularly methods that are suitable for use in microfluidic devices.
The discussion of prior publications and other prior knowledge does not constitute an admission that such material was published, known, or part of the common general knowledge.